The Environmental Protection Agency (EPA) and Organization for Economic Cooperation and Development (OECD) estimates that as many as 87,000 endocrine disruptor compounds (EDC) may be evaluated for estrogenic or anti-estrogenic activity. The development of standardized protocols to test for hormonally active EDC compounds is a major goal of regulatory agencies worldwide. Also, the role of phytoestrogens present in commercially available rodent diets on reproduction and carcinogenicity endpoints when EDCs are being evaluated requires serious consideration. Our studies were aimed at determining the effects of dietary factors including the phytoestrogen content (daidzein and genistein) and total metabolizable energy (ME) on uterine weights and vaginal opening (VO) endpoints of immature mice used in uterotrophic assays. We also assessed VO as an endpoint for selecting the most appropriate diets(s) for comparing the estrogenic or anti-estrogenic activity of EDCs. Pre-pubertal CD-1 mice were weaned at post-natal day (PND) 15 and fed test diets containing predetermined levels of dietary estrogens from PND 15 to PND 22 to determine the effects of dietary phytoestrogens on uterine weights. Mice were fed the test diets from PND 15 to PND 31 to determine the effects of dietary phytoestrogens on the time of VO. Vaginal opening was recorded daily from PND 20 to PND 31.The phytoestrogen content of the diet was highly predictive (P less than 0.0001) of the proportion of mice exhibiting VO at PND 24. Total ME was also significantly (P less than 0.01) correlated with the time of VO, although the predictability of this variable was less powerful than the phytoestrogen content. The time of VO in mice was significantly (P less than 0.05) accelerated in mice fed diets high in phytoestrogens as compared to low phytoestrogen content diets. It was concluded that 1) dietary daidzein and genistein can significantly (P less than 0.01) accelerate the time of VO in CD-1 mice, 2) the advancement in the time of VO was more highly correlated with the daidzein and genistein content of the diets than with the total ME, however, total ME was more highly correlated with the increase in uterine weights in immature mice than the daidzein and genistein content, 3) the advancement in the time of VO is a sensitive endpoint for evaluating the estrogenic activity of EDCs, and should be part of the standard protocol for evaluating EDCs, and 4) phytoestrogen-free diet(s) containing the same level of ME should be used in bioassays that compare the time of VO, or increases in uterine weights as endpoints for evaluating the estrogenic activity of an EDC.